Treatment of rhinitis

ABSTRACT

The invention provides a method of treating rhinitis. The method comprises administering an effective amount of a pharmaceutical composition comprising a diketopiperazine with amino acid side chains of aspartic acid and alanine (DA-DKP) formulated for nasal administration. The invention also provides a pharmaceutical product comprising a DA-DKP containing composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Patent Application No. 61/552,508, filed Oct. 28,2011 and U.S. Provisional Patent Application No. 61/561,215, filed Nov.17, 2011. The entire disclosures of each U.S. Provisional PatentApplications Nos. 61/552,508 and 61/561,215 are incorporated herein byreference.

FIELD OF INVENTION

The invention relates to a method of treating rhinitis. The methodcomprises administering an effective amount of a pharmaceuticalcomposition comprising a diketopiperazine with amino acid side chains ofaspartic acid and alanine (DA-DKP). The invention also provides apharmaceutical product comprising a DA-DKP containing composition.

BACKGROUND

Rhinitis is caused by chronic or acute inflammation of the mucousmembranes of the nose due to viruses, bacteria or irritants. Theinflammation results in the generation of excessive amounts of mucous,commonly producing a runny nose, nasal congestion and post-nasal drip.Rhinitis is reported to affect more than 50 million people in the UnitedStates alone.

There are several types of rhinitis, including infectious rhinitis,allergic rhinitis and nonallergic rhinitis. Infectious rhinitis iscaused by a viral or bacterial infection. Types of infectious rhinitisinclude the common cold and sinusitis.

Allergic rhinitis affects more than 20% of people worldwide and theprevalence increases annually. Allergic rhinitis causes impaired sociallife, sleep, school, and work. The quality of life of patients can bealtered by the severity and duration of rhinitis. Allergic rhinitis is aproinflammatory immune response to outdoor or indoor allergens, such asdust or pollen. Symptoms can occur year-round or primarily be at certaintimes of the year, usually in the spring, summer or fall. The AllergicRhinitis and its Impact on Asthma (ARIA) guidelines outlines themanagement of allergic rhinitis as allergen avoidance, patienteducation, pharmacotherapy, and allergen-specific immunotherapy. Forpharmacotherapy, ARIA currently recommends intranasal, second-generationH1-antihistamines and an intranasal corticosteroid for moderate tosevere persistent disease. See Bousquet et al., J. Allergy Clin.Immunol., 108 (Suppl 5):S147-334 (2001) and Bousquet et al., Allergy, 63(Suppl. 86):8-160 (2008).

Nonallergic rhinitis is rhinitis that is not triggered by allergens orinfectious agents. There is still much to be learned about nonallergicrhinitis, but it is thought that the triggers of it cause dilation ofthe blood vessels in the lining of the nose, which results in swellingand drainage. Types of nonallergic rhinitis include vasomotor,autonomic, hormonal, drug-induced, atrophic and gustatory rhinitis andrhinitis medicamentosa. Triggers of vasomotor rhinitis include smells,fumes, smoke, dust and temperature changes, and vasomotor rhinitis cancoexist with allergic rhinitis. Rhinitis medicamentosa is a condition ofrebound nasal congestion brought on by extended use of topicaldecongestants.

SUMMARY OF THE INVENTION

One embodiment of the invention relates to a method of treating rhinitisby administering an effective amount of a pharmaceutical compositioncomprising a diketopiperazine with amino acid side chains of asparticacid and alanine (DA-DKP), to an animal in need thereof. In one aspect,the rhinitis is allergic rhinitis.

In one aspect, the effective amount of the DA-DKP in the composition isfrom about 100 μg to about 3000 μg per day. In another aspect, theeffective amount of the DA-DKP in the composition is from about 500 μgto about 1500 μg per day. In yet other aspects, the administration ofthe composition comprising DA-DKP, is commenced within 24 hours ofdiagnosis of rhinitis. In still other aspects, the administration of thecomposition comprising DA-DKP, is commenced at the appearance of one ormore early signs of, or a predisposition to develop, rhinitis. One ormore early signs of rhinitis can be rhinorrhea, nasal congestion, nasalitching and sneezing. In yet other aspects of the method, the DA-DKP isin a composition prepared by removing albumin from a solution of a humanserum albumin composition. For example, the step of removing can be bytreating the human serum albumin composition by a separation method.Such separation methods can include ultrafiltration, sucrose gradientcentrifugation, chromatography, salt precipitation, and sonication. Inaddition, the step of removing can be by passing the human serum albumincomposition over an ultrafiltration membrane with a molecular weight cutoff that retains the albumin, and the resulting filtrate contains theDA-DKP. In one aspect, the ultrafiltration membrane has a molecularweight cutoff of less than 50 kDa. In still another aspect, theultrafiltration membrane has a molecular weight cut off less than 40kDa, less than 30 kDa, less than 20 kDa, less than 10 kDa, less than 5kDa or less than 3 kDa.

In yet another aspect, the composition comprising DA-DKP is administeredin combination with a second drug suitable for treating rhinitis. Forexample, the second drug can be selected from antihistamines,decongestants, anti-inflammatories, mast cell stabilizers, leukotrienemodifiers and IgE blockers.

Another embodiment of the invention relates to a pharmaceutical product,comprising a DA-DKP containing composition formulated for administrationby a route selected from inhalation, insufflation and nasaladministration to the nose. In one aspect, the DA-DKP containingcomposition formulated for administration by inhalation is packaged in adevice selected from insufflators, nebulizers, pressurized packs,squeeze bottle, a syringe, a dropper, a spray device, an atomizerdevice, and an aerosolizer. In one aspect, the pressurized pack cancomprise a propellant selected from dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide.

In still another aspect of the pharmaceutical product, the DA-DKPcontaining composition formulated for administration by inhalation orinsufflation comprises a powder mix of the DA-DKP containing compositionand a powder base. In one aspect, the powder mix can be in a dosage formselected from capsules, cartridges, gelatin packs and blister packs. Thepowder mix can be delivered by a device selected from an inhalator,insufflator and metered-dose inhaler.

In yet another aspect of the pharmaceutical product, the DA-DKPcontaining composition formulated for nasal administration is in a formof drops or sprays. In one aspect, the DA-DKP in the DA-DKP containingcomposition comprises from about 0.1% (w/v) to about 10% (w/v) of thecomposition. The drops or sprays can be contained within an intranasaldelivery system. In one aspect, the intranasal delivery system comprisesan atomizing device. In one aspect, the atomizing device comprises abottle and pump. In a preferred aspect, the pump is a metered dose pump.The metered dose pump can deliver an intranasal volume of the DA-DKPcontaining composition. In one aspect, the metered dose pump can deliveran intranasal volume of about 0.15 ml of the DA-DKP containingcomposition per pump. In still another pharmaceutical product furthercomprises an aqueous or non-aqueous base comprising one or more agentsselected from dispersing agents, solubilizing agents, and suspendingagents.

In still another aspect of the pharmaceutical product, the DA-DKPcontaining composition is formulated for nasal administration in a formselected from ointments, gels and creams. In one aspect, pharmaceuticalproduct further comprises excipients selected from animal fats,vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycol, silicones, bentonites, silicic acid,talc, zinc oxide and mixtures thereof. In still another aspect, thepharmaceutical product further comprises an absorption or permeationenhancer. In yet another aspect, the pharmaceutical product furthercomprises a thickening agent or viscosity enhancer to increase theresidence time of the DA-DKP containing composition in the nose. Instill another aspect, the pharmaceutical product further comprises apharmaceutically-acceptable carrier.

In yet other aspects of the pharmaceutical product, the DA-DKP is in acomposition prepared by removing albumin from a solution of a humanserum albumin composition. For example, the step of removing can be bytreating the human serum albumin composition by a separation method.Such separation methods can include ultrafiltration, sucrose gradientcentrifugation, chromatography, salt precipitation, and sonication. Inaddition, the step of removing can be by passing the human serum albumincomposition over an ultrafiltration membrane with a molecular weight cutoff that retains the albumin, and the resulting filtrate contains theDA-DKP. In one aspect, the ultrafiltration membrane has a molecularweight cutoff of less than 50 kDa. In still another aspect, theultrafiltration membrane has a molecular weight cut off less than 40kDa, less than 30 kDa, less than 20 kDa, less than 10 kDa, less than 5kDa or less than 3 kDa.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for a method of treating rhinitis, including butnot limited to infectious rhinitis, allergic rhinitis and nonallergicrhinitis. The method comprises administering to an animal in needthereof an effective amount of a pharmaceutical composition comprisingDA-DKP.

The invention also provides a pharmaceutical product formulated foradministration by a route such as inhalation, insufflation or nasaladministration to the nose. The composition comprises a DA-DKPcontaining composition.

Allergic rhinitis is a proinflammatory immune response to outdoor orindoor allergens, such as dust or pollen. Nonallergic rhinitis isrhinitis that is not triggered by allergens or infectious agents. Typesof nonallergic rhinitis include but are not limited to vasomotor,autonomic, hormonal, drug-induced, atrophic and gustatory rhinitis andrhinitis medicamentosa.

In order to treat rhinitis, the composition comprising DA-DKP isadministered to an animal in need of treatment. Preferably, the animalis a mammal, such as a rabbit, goat, dog, cat, horse or human. Mostpreferably, the animal is a human.

Effective dosage forms, modes of administration and dosage amounts forthe compounds of the invention (i.e., a composition comprising DA-DKP,and preparations comprising a filtrate of DA-DKP, such as a <5000MWfraction as discussed below) may be determined empirically using theguidance provided herein. It is understood by those skilled in the artthat the dosage amount will vary with the particular disease orcondition to be treated, the severity of the disease or condition, theroute(s) of administration, the duration of the treatment, the identityof any other drugs being administered to the animal, the age, size andspecies of the animal, and like factors known in the medical andveterinary arts. In general, a suitable daily dose of a compound of thepresent invention will be that amount of the compound which is thelowest dose effective to produce a therapeutic effect. However, thedaily dosage will be determined by an attending physician orveterinarian within the scope of sound medical judgment. If desired, theeffective daily dose may be administered as two, three, four, five, sixor more sub-doses, administered separately at appropriate intervalsthroughout the day. Administration of the compound should be continueduntil an acceptable response is achieved. Such an acceptable responsemay be for example when the symptoms of rhinitis are reduced and/or whenthe symptoms of rhinitis are no longer detected by the subject.

The composition of the present invention may be a pharmaceuticalsolution having a DA-DKP concentration range with a lower endpoint ofabout 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110μM, about 120 μM, about 130 μM, about 140 μM, about 150 μM, about 160μM, about 170 μM, about 180 μM, about 190 μM, about 200 μM, about 210μM, about 220 μM, about 230 μM, about 240 μM, about 240, about 250 μM,about 260 μM, about 270 μM, about 280 μM, about 290 μM, about 300 μM,about 310, about 320 μM, about 330 μM, about 340 μM, about 350 μM, about360 μM, about 370 μM, about 380 μM, about 390 μM, or about 400 μM. Thecomposition of the present invention may be a pharmaceutical solutionhaving a DA-DKP concentration range with an upper endpoint of about 600μM, about 580 μM, about 570 μM, about 560 μM, about 550 μM, about 540μM, about 530 μM, about 520 μM, about 510 μM, about 500 μM, about 490μM, about 480 μM, about 470 μM, about 460 μM, about 450 μM, about 440μM, about 430 μM, about 420 μM, about 410 μM, about 400 μM, about 390μM, about 380 μM, about 370 μM, about 360 μM, about 350, about 340 μM,about 330 μM, about 320 μM, about 310 μM, about 300 μM, about 290 μM,about 280, about 270 μM, about 260 μM, about 250 μM, about 240 μM, about230 μM, about 220 μM, about 210 μM, or about 200 μM.

An effective amount of DA-DKP in the composition of the presentinvention for treating rhinitis can be a range with a lower endpoint ofabout 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg,about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 85 μg, about90 μg, about 95 μg, about 100 μg, about 110 μg, about 120 μg, about 130μg, about 140 μg, about 150 μg, about 160 μg, about 170 μg, about 180μg, about 190 μg, about 200 μg, about 210 μg, about 220 μg, about 230μg, about 240 μg, about 250 μg, about 260 μg, about 270 μg, about 280μg, about 290 μg, about 300 μg, about 310 μg, about 320 μg, about 330μg, about 340 μg, about 350 μg, about 360 μg, about 370 μg, about 380μg, about 390 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μgor about 500 μg. An effective amount of DA-DKP in the composition of thepresent invention for treating rhinitis can be a range with upperendpoint of about 500 μg, about 490 μg, about 480 μg, about 470 μg,about 460 μg, about 450 μg, about 440 μg, about 430 μg, about 420 μg,about 410 μg, about 400 μg, about 390 μg, about 380 μg, about 370 μg,about 360 μg, about 350 μg, about 340 μg, about 330 μg, about 320 μg,about 310 μg, about 300 μg, about 290 μg, about 280 μg, about 270 μg,about 260 μg, about 250 μg, about 240 μg, about 230 μg, about 220 μg,about 210 μg, about 200 μg, about 190 μg, about 180 μg, about 170 μg,about 160 μg, about 150 μg, about 140 μg, about 130 μg, about 120 μg,about 110 μg, about 100 μg, about 90 μg, about 80 μg, about 70 μg, about60 μg, about 50 μg, about 40 μg, about 30 μg, or about 20 μg.

Additionally, when the DA-DKP containing composition is administered asa spray composition or formulation and/or as drop composition orformulation, the effective dosage amounts can be a range with a lowerend point of about 0.01% (weight/volume (w/v)), about 0.02% (w/v), about0.03% (w/v), about 0.04% (w/v), about 0.05% (w/v), about 0.06% (w/v),about 0.07% (w/v), about 0.08% (w/v), about 0.09% (w/v), about 0.10%(w/v), about 0.11% (w/v), about 0.12% (w/v), about 0.13% (w/v), about0.14% (w/v), about 0.15% (w/v), about 0.16% (w/v), about 0.17% (w/v),about 0.18% (w/v), about 0.19% (w/v), about 0.20% (w/v), about 0.25%(w/v), about 0.30% (w/v), about 0.35% (w/v), about 0.40% (w/v), about0.45% (w/v), about 0.50% (w/v). Most preferably about 0.1% (w/v). Also,when the DA-DKP containing composition is administered as a spraycomposition or formulation and/or as drop composition or formulation,the effective dosage amounts can be a range with a upper end point about10.0% (w/v), about 9.0% (w/v), about 8.0% (w/v), about 7.0% (w/v), about6.0% (w/v), about 5.0% (w/v), about 4.0% (w/v), about 3.0% (w/v), about2.0% (w/v), 1.0% (w/v), about 0.95% (w/v), about 0.90% (w/v), about0.85% (w/v), about 0.80% (w/v), about 0.75% (w/v), about 0.70% (w/v),about 0.65% (w/v), about 0.60% (w/v), or about 0.55% (w/v).

The administration of the DA-DKP containing composition may be commencedwithin 24 hours of diagnosis of rhinitis. The administration of theDA-DKP containing composition may be commenced at the appearance of oneor more early signs of, or a predisposition to develop, rhinitis. Theearly signs of rhinitis include but are not limited torhinorrhea, nasalcongestion, nasal itching and sneezing.

The compounds of the present invention (i.e., a composition comprisingDA-DKP, and preparations comprising a filtrate of DA-DKP, such as a<5000MW fraction as discussed below) may be administered to an animalpatient for therapy by any suitable route of administration, includingorally, nasally, parenterally (e.g., intravenously, intraperitoneally,subcutaneously or intramuscularly), transdermally, intraocularly andtopically (including buccally and sublingually). Preferred is oral,ocular or nasal administration for any disease or condition treatableaccording to the invention. Especially preferred is nasaladministration.

While it is possible for a DA-DKP containing composition of the presentinvention to be administered alone, it is preferable to administer theDA-DKP containing composition as a pharmaceutical formulation orproduct. The pharmaceutical compositions of the invention comprise acompound or compounds of the invention as an active ingredient inadmixture with one or more pharmaceutically-acceptable carriers and,optionally, with one or more other compounds, drugs or other materials.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to theanimal. Pharmaceutically-acceptable carriers are well known in the art.Regardless of the route of administration selected, the DA-DKPcontaining compositions of the present invention are formulated intopharmaceutically-acceptable dosage forms by conventional methods knownto those of skill in the art. See, e.g., Remington's PharmaceuticalSciences.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules or as asolution or a suspension in an aqueous or non-aqueous liquid, or anoil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), and the like, each containing a predeterminedamount of a compound or compounds of the present invention as an activeingredient. Formulations of the present invention may also beadministered as bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient (i.e., a composition comprising DA-DKP, andpreparations comprising a filtrate of DA-DKP, such as a <5000MW fractionas discussed below) is mixed with one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate,and/or any of the following: (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders,such as, for example, carboxymethylcellulose, alginates, gelatin,polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such asglycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonosterate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may be employed as fillers in soft andhard-filled gelatin capsules using such excipients as lactose or milksugars, as well as high molecular weight polyethylene glycols and thelike.

A tablet may be made by compression or molding optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active ingredient, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Pharmaceutical formulations and products include those suitable foradministration by inhalation or insufflation or for nasaladministration. For administration to the upper (nasal) or lowerrespiratory tract by inhalation, the DA-DKP containing compositions ofthe invention are conveniently delivered from a device for inhalationdelivery such as an insufflator, nebulizer or a pressurized pack orother convenient means of delivering an aerosol spray. Pressurized packsmay comprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecomposition may take the form of a dry powder, for example, a powder mixof the DA-DKP containing composition of the invention and a suitablepowder base, such as lactose or starch. The powder composition may bepresented in unit dosage form in, for example, capsules or cartridges,or, e.g., gelatin or blister packs from which the powder may beadministered with the aid of an inhalator, insufflator or a metered-doseinhaler.

For nasal administration, DA-DKP containing compositions of theinvention may be administered by means of nose drops or a liquid spray,such as by means of a plastic bottle spray or atomizer or metered-doseinhaler. Liquid sprays are conveniently delivered from pressurizedpacks.

Nose drops may be formulated with an aqueous or nonaqueous base alsocomprising one or more dispersing agents, solubilizing agents orsuspending agents. Drops can be delivered by means of a simple eyedropper-capped bottle or by means of a plastic bottle adapted to deliverliquid contents dropwise by means of a specially shaped closure.

Ointments, gels and creams can also be used for nasal administration ofthe DA-DKP containing compositions of the invention. The activeingredient may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required. The ointments, creams and gels maycontain, in addition to the active ingredient, excipients, such asanimal and vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Dosage forms for topical administration or for transdermaladministration of compounds of the invention include powders, sprays,ointments, pastes, creams, lotions, gels, solutions, patches, drops andinhalants. The active ingredient may be mixed under sterile conditionswith a pharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required. The ointments, pastes, creams andgels may contain, in addition to the active ingredient, excipients, suchas animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.Powders and sprays can contain, in addition to the active ingredient,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder or mixtures of these substances.Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane. Transdermal patches have the added advantage ofproviding controlled delivery of compounds of the invention to the body.Such dosage forms can be made by dissolving, dispersing or otherwiseincorporating one or more compounds of the invention in a proper medium,such as an elastomeric matrix material. Absorption enhancers can also beused to increase the flux of the compound across the skin. The rate ofsuch flux can be controlled by either providing a rate-controllingmembrane or dispersing the compound in a polymer matrix or gel. Adrug-impregnated solid carrier (e.g., a dressing) can also be used fortopical administration.

Pharmaceutical compositions of this invention suitable for parenteraladministrations comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents. Also, drug-coated stents may be used.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drug isaccomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

While it is possible for a DA-DKP containing composition to be givenalone to treat rhinitis, alternatively, the DA-DKP containingcomposition may be given in combination with one or more othertreatments or drugs suitable for treating the rhinitis. For instance,the DA-DKP containing composition can be administered prior to, inconjunction with (including simultaneously with), or after the othertreatment or drug. In the case of another drug, the drug and the DA-DKPcontaining composition may be administered in separate pharmaceuticalcompositions or as part of the same pharmaceutical composition. Suitableother drugs include antihistamines, decongestants, anti-inflammatories(steroidal and nonsteroidal), mast cell stabilizers, leukotrienemodifiers and IgE blockers. Specific suitable drugs includefexofenadine, doxylamine, diphenhydramine, triprolidine, loratidine,cetirizine, pseudophedrine, phenylephrine, aspirin, ibuprofen, naproxen,prednisone, prednisolone, and methylprednisolone. Suitable drugs forinclusion in a nasal spray are steroids (such as fluticasone propionate,mometasone, budesonite, flunisolide, triamcinolone and beclomethasone),antihistamines (such as azelastine), anticholinergics (such asipratproium) and mast cell stabilizers (such as cromolyn).

Methods of making diketopiperazines, such as DA-DKP, are well known inthe art, and these methods may be employed to synthesize thediketopiperazines of the invention. See, e.g., U.S. Pat. Nos. 4,694,081,5,817,751, 5,990,112, 5,932,579, 6,555,543; US Patent ApplicationPublication Number 2004/0024180, PCT applications WO 96/00391 and WO97/48685, and Smith et al., Bioorg. Med. Chem. Letters, 8, 2369-2374(1998), the complete disclosures of which are incorporated herein byreference.

For instance, diketopiperazines, such as DA-DKP, can be prepared byfirst synthesizing dipeptides. The dipeptides can be synthesized bymethods well known in the art using L-amino acids, D-amino acids or acombination of D- and L-amino acids.

Preferred are solid-phase peptide synthetic methods. Of course,dipeptides are also available commercially from numerous sources,including DMI Synthesis Ltd., Cardiff, UK (custom synthesis),Sigma-Aldrich, St. Louis, Mo. (primarily custom synthesis), PhoenixPharmaceuticals, Inc., Belmont, Calif. (custom synthesis), FisherScientific (custom synthesis) and Advanced ChemTech, Louisville, Ky.

Once the dipeptide is synthesized or purchased, it is cyclized to form adiketopiperazine. This can be accomplished by a variety of techniques.For example, U.S. Patent Application Publication Number 2004/0024180describes a method of cyclizing dipeptides. Briefly, the dipeptide isheated in an organic solvent while removing water by distillation.Preferably, the organic solvent is a low-boiling azeotrope with water,such as acetonitrile, allyl alcohol, benzene, benzyl alcohol, n-butanol,2-butanol, t-butanol, acetic acid butylester, carbon tetrachloride,chlorobenzene chloroform, cyclohexane, 1,2-dichlorethane, diethylacetal,dimethylacetal, acetic acid ethylester, heptane, methylisobutylketone,3-pentanol, toluene and xylene. The temperature depends on the reactionspeed at which the cyclization takes place and on the type ofazeotroping agent used. The reaction is preferably carried out at50-200° C., more preferably 80-150° C. The pH range in which cyclizationtakes place can be easily determine by the person skilled in the art. Itwill advantageously be 2-9, preferably 3-7.

When one or both of the amino acids of the dipeptide has, or isderivatized to have, a carboxyl group on its side chain (e.g., asparticacid or glutamic acid), the dipeptide is preferably cyclized asdescribed in U.S. Pat. No. 6,555,543. Briefly, the dipeptide, with theside-chain carboxyl still protected, is heated under neutral conditions.Typically, the dipeptide will be heated at from about 80° C. to about180° C., preferably at about 120° C. The solvent will be a neutralsolvent. For instance, the solvent may comprise an alcohol (such asbutanol, methanol, ethanol, and higher alcohols, but not phenol) and anazeotropic co-solvent (such as toluene, benzene, or xylene). Preferably,the alcohol is butan-2-ol, and the azeotropic co-solvent is toluene. Theheating is continued until the reaction is complete, and such times canbe determined empirically. Typically, the dipeptide will be cyclized byrefluxing it for about 8-24 hours, preferably about 18 hours. Finally,the protecting group is removed from the diketopiperazine. In doing so,the use of strong acids (mineral acids, such as sulfuric or hydrochloricacids), strong bases (alkaline bases, such as potassium hydroxide orsodium hydroxide), and strong reducing agents (e.g., lithium aluminumhydride) should be avoided, in order to maintain the chirality of thefinal compound.

Dipeptides made on solid phase resins can be cyclized and released fromthe resin in one step. See, e.g., U.S. Pat. No. 5,817,751. For instance,the resin having an N-alkylated dipeptide attached is suspended intoluene or tolueneethanol in the presence of acetic acid (e.g., 1%) ortriethylamine (e.g., 4%). Typically, basic cyclization conditions arepreferred for their faster cyclization times.

Other methods of cyclizing dipeptides and of making diketopiperazinesare known in the art and can be used in the preparation ofdiketopiperazines useful in the practice of the invention. See, e.g.,those references listed above. In addition, many diketopiperazinessuitable for use in the present invention can be made as described belowfrom proteins and peptides. Further, diketopiperazines for use in thepractice of the invention can be obtained commercially from, e.g., DMISynthesis Ltd., Cardiff, UK (custom synthesis).

The DA-DKP composition and/or products of the present invention can beprepared from solutions containing DA-DKP, including from thecommercially-available pharmaceutical compositions comprising albumin,such as human serum albumin, by well known methods, such asultrafiltration, chromatography (size-exclusion chromatography (e.g.,Centricon filtration), affinity chromatography (e.g., using a column ofbeads having attached thereto an antibody or antibodies directed to thedesired diketopiperazine(s) or an antibody or antibodies directed to thetruncated protein or peptide), anion exchange or cation exchange),sucrose gradient centrifugation, chromatography, salt precipitation, orsonication, that will remove some or all of the albumin in the solution.The resultant DA-DKP-containing composition and/or product can be usedand incorporated into pharmaceutical compositions as described above.

Using an ultrafilration separation method, a human serum albumincomposition can be passed over an ultrafiltration membrane having amolecular weight cut-off that retains the albumin while the DA-DKPpasses into the resulting filtrate or fraction. This filtrate maycomprise components having molecular weights less than about 50 kDA,less than about 40 kDa, less than 30 kDa, less than about 20 kDa, lessthan about 10 kDa, less than about 5 kDa, less than about 3 kDa.Preferably, the filtrate comprises components having molecular weightsless than about 5 kDa (also referred to as “<5000MW”). This <5000MWfraction or filtrate contains DA-DKP which is formed after the dipeptideaspartate-alanine is cleaved from albumin and subsequently cyclized intothe diketopiperazine. The <5000MW Fraction can be prepared as describedin U.S. Pat. No. 7,732,403, the complete disclosure of which isincorporated herein by reference.

Physiologically-acceptable salts of the DA-DKP of the invention may alsobe used in the practice of the invention. Physiologically-acceptablesalts include conventional non-toxic salts, such as salts derived frominorganic acids (such as hydrochloric, hydrobromic, sulfuric,phosphoric, nitric, and the like), organic acids (such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,glutamic, aspartic, benzoic, salicylic, oxalic, ascorbic acid, and thelike) or bases (such as the hydroxide, carbonate or bicarbonate of apharmaceutically-acceptable metal cation or organic cations derived fromN,N-dibenzylethylenediamine, D-glucosamine, or ethylenediamine). Thesalts are prepared in a conventional manner, e.g., by neutralizing thefree base form of the compound with an acid.

Kits comprising the pharmaceutical products of the present invention arealso provided. The kits can comprise a DA-DKP composition formulated fornasal administration. The DA-DKP can be prepared as described herein,such as by removing albumin from a solution of a human albumincomposition. The kits may contain unit-dose or multi-dose sealedcontainers, for example, squeeze bottles, syringes, a dropper, a spraydevice, an atomizer device, an aerosolize, a nebulizer, an insufflatorsor pressurized packs. The kits may be stored in a condition, wherein thecontents are ready for direct use. Alternately, the kits may be storedin a lyophilized condition requiring only the addition of the sterileliquid carrier, for example water, immediately prior to use.

As used herein, “a” or “an” means one or more.

As used herein, “comprises” and “comprising” include within their scopeall narrower terms, such as “consisting essentially of” and “consistingof” as alternative embodiments of the present invention characterizedherein by “comprises” or “comprising”. In regard to use of “consistingessentially of”, this phrase limits the scope of a claim to thespecified steps and materials and those that do not materially affectthe basic and novel characteristics of the invention disclosed herein.

Additional objects, advantages and novel features of the presentinvention will become apparent to those skilled in the art byconsideration of the following non-limiting examples. The followingexperimental results are provided for purposes of illustration and arenot intended to limit the scope of the invention.

EXAMPLE

A Phase Ib randomized, double-blinded, placebo-controlled, parallelgroup study was performed to evaluate the efficacy of the <5000MWFraction for treating allergic rhinitis in adult humans. Briefly, thestudy was performed as follows.

The subjects were male or female humans, 18-65 years of age, with ahistory of allergic rhinitis that had been receiving therapy (continuousor intermittent) for more than 1 year. Each subject had a demonstratedsensitivity to at least one seasonal allergen (treegrass pollen) knownto induce allergic rhinitis through a standard skin test. Each subjecthad a minimum subject-reported reflective Total Nasal Symptom Score(rTNSS) of ≧5 on the day of the Screening Visit (day 1).

During a seven-day wash out period (days 1-7), the subjects received notreatment of any type and recorded their symptoms twice a day (in themorning before bathing, consumption of food or beverages or strenuousactivities and 12 hours later) using the following scale:

-   -   0=absent (no sign/symptom present)    -   1=mild (sign/symptom clearly present, but minimal awareness;        easily tolerated)    -   2=moderate (definite awareness of sign/symptom that is        bothersome but tolerable)    -   3=severe (sign/symptom that is hard to tolerate; causes        interference with activities of daily living and/or sleeping)

The subjects were randomized into treatment groups (<5000MW Fraction orplacebo (i.e. saline)) after the wash out period (on day 8).

Treatment was begun on day 8 and continued through day 17 (10 daystotal). During treatment, the subjects again recorded their symptomstwice daily (as described above and before administration of the testmedications in the morning) using the above scale. The subjectsadministered 0.3 ml of one of the test medications, 0.15 ml per nostril,once daily in the morning, immediately after recording their symptoms.The first administration of test medications was supervised by test sitepersonnel. The test medications were the <5000MW Fraction (containing3.72% DA-DKP) used as a nasal spray and placebo spray (containing normalsaline, 0.9% NaCl) in identical intranasal spray format.

Efficacy of the Test Medications was Assessed as Follows:

The measures of effectiveness in this study included thesubject-reported Total Nasal Symptom Score (TNSS). The TNSS is definedas the sum of the subject-reported symptom scores for the four nasalsymptoms: rhinorrhea (runny nose), nasal congestion, nasal itching, andsneezing. Each score is assessed on a severity scale ranging from 0 to 3as defined above.

The subjects were asked to assess both reflective TNSS (i.e., anevaluation of symptom severity over the past 12 hours prior to therecording of the score) and instantaneous TNSS (i.e., an evaluation ofthe symptom severity over the last 10 minutes).

The reflective and instantaneous TNSS are defined as the sum of thesubject-reported symptom scores for the four nasal symptoms. Eachsubject recorded the symptom scores in the subject's diary. For eachscore, information recorded in the diary included the following:

-   -   rhinorrhea (runny nose)    -   nasal congestion    -   nasal itching    -   sneezing        The severity scale for each symptom evaluation is given above.

Mean reflective (r) and instantaneous (i) subject-reported total nasalsymptom scores (TNSS) were calculated for each subject. The mean TNSS isthe average of all AM and PM daily scores (each score is ranked on ascale from 0-12) during the baseline (baseline efficacy value) and thetreatment period (double-blind efficacy value).

The change in efficacy was calculated as the change in the double-blindvalue—baseline value as follows:

-   -   Change in rTNSS: mean double-blind rTNSS—mean baseline rTNSS    -   Change in iTNSS: mean double-blind iTNSS—mean baseline iTNSS

Student's t-test: mean (SD) difference between treatment groups for thefollowing:

-   -   Mean change in (rTNSS/iTNSS)    -   Mean change in AM (rTNSS/iTNSS)    -   Mean change in PM (rTNSS/iTNSS)    -   Mean change in individual symptom scores for each of the four        nasal symptoms.

The results obtained by analysis using the Student's t-test are providedin Tables 1-5 and are summarized below (number of subjects who completedthe trial at the time of the interim analysis: n=20; placebo, n=10(Intent to Treat (ITT) population; >5000MW Fraction, n=10 (ITTpopulation).

rTNSS: <5000MW Fraction showed a 2.4 point decrease in reflectivesymptom severity compared to 1.7 point decrease with placebo (Table 2).There was 35% improvement in rTNSS with <5000MW Fraction versus 23%improvement with placebo. The largest improvements were with sneezingand runny nose symptoms (Table 3). 6/7 efficacy measures showed a largerimprovement with <5000MW Fraction than placebo (all assessments butitchy nose symptom). There was significant rTNSS improvement in subsetwith baseline rTNSS <median (7.5).

TABLE 1 Baseline efficacy variables, mean (SD) <5000 MW EfficacyVariable Placebo Fraction T-test p value rTNSS 7.46 (1.71) 6.84 (1.39)0.39 iTNSS 7.40 (1.71) 6.45 (1.11) 0.16 PNSS (screening) 6.50 (1.43)5.60 (1.65) 0.21 PNSS 6.90 (1.60) 5.50 (0.85) 0.03 (randomization) PNSS= Physician Nasal Symptoms Score; TNSS = Total Nasal Symptoms Score

TABLE 2 Mean change in rTNSS Efficacy Saline n = 10 <5000 MW VariableMean (SD) Fraction n = 10 (SD) T-test p value rTNSS −1.71 (1.82) −2.37(2.06) 0.46 AM rTNSS −1.68 (1.69) −2.25 (2.00) 0.51 PM rTNSS −1.72(1.95) −2.03 (2.08) 0.74

TABLE 3 Mean Change in Reflective Symptoms Saline n = 10 <5000 MWSymptom Mean (SD) Fraction n = 10 (SD) T-test p value rRunny nose −0.33(0.8) −0.60 (0.8) 0.45 rStuffy nose −0.58 (0.6) −0.59 (0.5) 0.97 rItchynose −0.54 (0.6) −0.53 (0.6) 0.97 rSneezing −0.26 (0.3) −0.65 (0.7) 0.13

iTNSS: <5000MW Fraction showed a 2.0 point decrease in instantaneoussymptom severity compared to 1.7 point decrease with placebo (Table 4).57 efficacy measures showed a larger improvement with <5000MW Fractionthan with placebo. The largest improvements were with sneezing nosesymptoms (Table 5). Smaller improvements were seen with instantaneousversus reflective symptoms.

TABLE 4 Mean Change in iTNSS Efficacy Saline n = 10 <5000 MW variableMean (SD) Fraction n = 10 (SD) T-test p value iTNSS −1.68 (1.84) −1.97(2.06) 0.75 AM iTNSS −1.55 (1.69) −1.84 (1.94) 0.73 PM iTNSS −1.50(1.61) −1.56 (1.98) 0.94

TABLE 5 Mean Change in Instantaneous Symptoms Placebo <5000 MW SymptomMean (SD) Fraction (SD) T-test p value iRunny nose −0.27 (0.8) −0.37(0.8) 0.78 iStuffy nose −0.54 (0.4) −0.53 (0.6) 0.98 iItchy nose −0.63(0.6) −0.46 (0.6) 0.53 iSneezing −0.24 (0.6) −0.61 (0.5) 0.16

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing exemplary claims.

What is claimed:
 1. A method of treating rhinitis comprisingadministering an effective amount of a pharmaceutical compositioncomprising a diketopiperazine with amino acid side chains of asparticacid and alanine (DA-DKP), to an animal in need thereof.
 2. The methodof claim 1, wherein the rhinitis is allergic rhinitis.
 3. The method ofclaim 1, wherein the effective amount of the DA-DKP in the compositionis from about 100 μg to about 3000 μg per day.
 4. The method of claim 1,wherein the effective amount of the DA-DKP in the composition is fromabout from about 500 μg to about 1500 μg per day.
 5. The method of claim1, wherein administration of the composition comprising DA-DKP, iscommenced within 24 hours of diagnosis of rhinitis.
 6. The method ofclaim 1, wherein administration of the composition comprising DA-DKP, iscommenced at the appearance of one or more early signs of, or apredisposition to develop, rhinitis.
 7. The method of claim 6, whereinthe one or more early signs of rhinitis are selected from the groupconsisting of rhinorrhea, nasal congestion, nasal itching and sneezing.8. The method of claim 1, wherein the DA-DKP is in a compositionprepared by removing albumin from a solution of a human serum albumincomposition.
 9. The method of claim 8, wherein the step of removing thealbumin comprises treating the human serum albumin composition by aseparation method selected from the group consisting of ultrafiltration,sucrose gradient centrifugation, chromatography, salt precipitation, andsonication.
 10. The method of claim 9, wherein the step of removingcomprises passing the human serum albumin composition over anultarfiltration membrane with a molecular weight cut off that retainsthe albumin, and wherein the resulting filtrate comprises DA-DKP. 11.The method of claim 10, wherein the ultrafiltration membrane hasmolecular weight cutoff of less than 50 kDa, less than 40 kDa, less than30 kDA, less than 20 kDa, less than 10 kDa, less than 5 kDa or less than3 kDA.
 12. The method of claim 1, wherein the composition comprisingDA-DKP is administered in combination with a second drug suitable fortreating rhinitis.
 13. The method of claim 12, wherein the second drugsuitable for treating rhinitis is selected from the group consisting ofantihistamines, decongestants, anti-inflammatories, mast cellstabilizers, leukotriene modifiers and IgE blockers.
 14. Apharmaceutical product, comprising a DA-DKP containing compositionformulated for administration by a route selected from the groupconsisting of inhalation, insufflation and nasal administration to thenose.
 15. The pharmaceutical product of claim 14, wherein the DA-DKPcontaining composition is formulated for administration by inhalation ispackaged in a device selected from the group consisting of insufflators,nebulizers, pressurized packs, squeeze bottle, a syringe, a dropper, aspray device, an atomizer device, and an aerosolizer.
 16. Thepharmaceutical product of claim 15, wherein the pressurized packcomprises a propellant selected from the group consisting ofdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, and carbon dioxide.
 17. The pharmaceuticalproduct of claim 14, wherein the DA-DKP containing compositionformulated for administration by inhalation or insufflation comprises apowder mix of the DA-DKP containing composition and a powder base. 18.The pharmaceutical product of claim 17, wherein the powder mix is in adosage form selected from the group consisting of capsules, cartridges,gelatin packs and blister packs.
 19. The pharmaceutical product of claim18, wherein the powder mix is delivered by a device selected from thegroup consisting of an inhalator, insufflator and metered-dose inhaler.20. The pharmaceutical product of claim 14, wherein the DA-DKPcontaining composition formulated for nasal administration is in a formof drops or sprays.
 21. The pharmaceutical product of claim 20, whereinthe DA-DKP comprises from about 0.1% (w/v) to about 10% (w/v) of thecomposition.
 22. The pharmaceutical product of claim 20, wherein thedrops or sprays are contained within an intranasal delivery system. 23.The pharmaceutical product of claim 22, wherein the intranasal deliverysystem comprises an atomizing device.
 24. The pharmaceutical product ofclaim 23, wherein the atomizing device comprises a bottle and a pump.25. The pharmaceutical product of claim 24, wherein the pump is ametered dose pump.
 26. The pharmaceutical product of claim 25, whereinthe metered dose pump delivers an intranasal volume of the DA-DKPcontaining composition of about 0.15 ml per pump.
 27. The pharmaceuticalproduct of claim 20, further comprising an aqueous or non-aqueous basecomprising one or more agents selected from the group consisting ofdispersing agents, solubilizing agents, and suspending agents.
 28. Thepharmaceutical product of claim 14, wherein the DA-DKP containingcomposition formulated for nasal administration is in a form selectedfrom the group consisting of ointments, gels and creams.
 29. Thepharmaceutical product of claim 28, wherein the DA-DKP containingcomposition further comprises excipients selected from the groupconsisting of animal fats, vegetable fats, oils, waxes, paraffins,starch, tragacanth, cellulose derivatives, polyethylene glycol,silicones, bentonites, silicic acid, talc, zinc oxide and mixturesthereof.
 30. The pharmaceutical product of claim 14, further comprisingan absorption or permeation enhancer.
 31. The pharmaceutical product ofclaim 14, further comprising a thickening agent or viscosity enhancer toincrease the residence time of the DA-DKP containing composition in thenose.
 32. The pharmaceutical product of claim 14, further comprising apharmaceutically-acceptable carrier.
 33. The pharmaceutical product ofclaim 14, wherein the DA-DKP is prepared by removing albumin from asolution of a human serum albumin composition.
 34. The pharmaceuticalproduct of claim 33, wherein the step of removing the albumin comprisestreating the human serum albumin composition by a separation methodselected from the group consisting of ultrafiltration, sucrose gradientcentrifugation, chromatography, salt precipitation, and sonication. 35.The pharmaceutical product of claim 34, wherein the step of removingcomprises passing the human serum albumin composition over anultarfiltration membrane with a molecular weight cut off that retainsthe albumin, and wherein the resulting filtrate comprises DA-DKP. 36.The pharmaceutical product of claim 35, wherein the ultrafiltrationmembrane has molecular weight cutoff of less than 50 kDa, less than 40kDa, less than 30 kDA, less than 20 kDa, less than 10 kDa, less than 5kDa or less than 3 kDA.